Biotin-peg-substrate for a lipase assay

ABSTRACT

Disclosed is a compound of Formula (I), wherein: L is a linking agent; B is a binding agent; X is an atom or group suitable for attaching L to the glycerol chain; and R is a straight chain saturated or unsaturated alkyl group having from 8 to 30 carbon atoms, substituted with M′ or M″ wherein at least one of M′ and/or M″ is a detectable label. The compound can be used as a lipase substrate in a solid phase-based assay system, such as a scintillation proximity assay, to detect lipase enzyme activity.

The present invention describes a novel substrate for use in an assayfor lipase enzyme activity. In particular, this novel substrate can belabelled and used in a homogeneous assay. INTRODUCTION

[0001] Lipases are enzymes that catalyse the hydrolysis oftriacylglycerols in the first step in recovering stored fatty acids forenergy production. The sequence of hydrolysis from the three positionson glycerol depends on the specificity of the particular lipaseinvolved.

[0002] Lipase enzyme activity is an important function and its strictregulation is necessary to ensure healthy metabolism. For example,lipases in adipose tissue are key enzymes for the release of majorenergy stores. Their activity is under hormonal control to ensure thattriacylglycerol hydrolysis is balanced with the process oftriacyglycerol synthesis to assure adequate energy stores and yet avoidlevels of fatty acids becoming so high as to cause adverse effects.

[0003] One example of a lipase is Hormone Sensitive Lipase (HSL). Thisenzyme catalyses the rate-limiting step in lipolysis and utilisation oftriacylglycerol stocks such as those found in adipose tissue andskeletal muscle. It is a cytosolic neutral lipase and itstriacylglycerol substrate is hydrophobic. The enzyme is activated ininsulin deficiency and/or insulin resistance causing depletion of lipidstocks in diabetes sufferers. Inhibition of the enzyme, therefore, isimportant in the management of diabetes.

[0004] Imbalances in the activity of lipases have also been linked toother clinical disorders including obesity and atherosclerotic vasculardisease. In view of the importance of lipase activity in disorders suchas these, it is necessary to have assays which are suitable formeasuring enzyme activity. Such assays could provide a useful diagnosisfor assessing the status of lipase activity-related disorders. Inaddition, assays that would be suitable for assessing the effectivenessof any compounds that may have the potential to modify (either toinhibit or enhance) enzyme activity are required.

[0005] Recent developments in genomics and combinatorial chemistry havegenerated large numbers of new drug targets and new compounds. Inconsequence, advances in drug discovery have focused on high throughputscreening (HTS) technologies and miniaturisation. These technologies aimto screen large numbers of either candidate drug targets or newcompounds to uncover new leads in a short time (High-throughputscreening for drug discovery, James R. Broach and Jeremy Thorner. NatureVol. 384, Supp, 7 November 1996, 14-16.). Key to the success of such HTStechniques is the development of assays which can be adapted into aminiaturised format and which have only a minimum number of steps inorder to facilitate an automated screening process.

[0006] As substrates for lipases are generally hydrophobic in nature,current methods for measuring lipase enzyme activity typically involve alarge number of processing steps including organic phase extraction.

[0007] For example, a typical current method for determining theactivity of a lipase, such as Hormone Sensitive Lipase (HSL), wouldinvolve a substrate such as triacylglycerol, cholesteryl ester ordiacylglycerol. The triacylglycerol substrate would, typically beradiolabelled e.g. by a 3H label. Prior to adding the substrate to theenzyme, a substrate emulsion would be formed by, for example, sonicatingor vortexing a solution of the substrate in a buffer solution (forexample 100 mM potassium phosphate at pH 7.0) containing 0.05% BSA or anemulsion stabiliser such as phospholipids. The substrate/enzyme mixturewould then be incubated at 37° C. for 60 minutes after which reactionswould be stopped by the addition of a stop solution (such aschloroform:methanol:heptane (25:23:18)). In order to determine enzymeactivity on the substrate, it would be necessary to separate theproducts of the enzyme reaction from the labelled substrate byextracting the organic phase of the reaction solution. This would beachieved by vortexing and centrifuging the tubes containing the reactionsolution and taking aliquots of the upper phase for liquid scintillationcounting. Results would be expressed in nmol of fatty acid released/mgprotein (see for example, Rapid assay for hormone-sensitive lipaseactivity of adipose tissue. H. Tomquist, L. Krabisch and P. Belfrage.Journal of Lipid Research,1972,13,424-426).

[0008] Such a multi-step procedure, involving vortexing andcentrifugation, would be difficult to automate and inconvenient toperform on a large number of samples simultaneously. Accordingly, todate, no HTS techniques have been made available for screening forlipase enzyme activity or for compounds which may act as enhancers orinhibitors of this activity.

[0009] Recently, assays based on homogeneous techniques have beendeveloped and these are readily adaptable to HTS. Generally, homogeneousassays involve detection via a solid phase binding step. One example ofsuch an assay is the Scintillation Proximity Assay (SPA) (Scintillationproximity assay—a versatile high throughput screening technology. Cook,N. D.; Drug Discovery Today, 1:287-294 (1996)). This assay involvessolid phase binding to microspheres or “beads” that contain ascintillant. If SPA beads are coated, for example, with a receptor andincubated with a ³H-labelled ligand, any ligand which binds to thereceptor will be brought in close proximity to the bead such that itsradiolabel excites the scintillant causing the emission of a lightsignal. SPA results can be read in 96 and 384 well plates using standardmicroplate scintillation counters or in 384 and higher density wellplates using LEADseeker™ Homogeneous Imaging System (Imaging ProximityAssays—The LEADseeker Homogeneous Imaging System, A. Fowler, M. Harvey,A. Cox, B. Jessop, M. Looker, I. Davis, J. Morris, A. Santos, J. Turnerand M. Price-Jones. Genetic Engineering News, Volume 18, Number 20, Nov.15, 1998). The LEADseeker™ system features a CCD camera allowing imagingof high density microtitre plates in a single pass.

[0010] However, in order to adapt any assay which depends on solid phasebinding, such as SPA, to an assay for lipase enzyme activity, it wouldbe necessary to modify the lipase substrate to render it capable ofbinding to a surface. Due to their hydrophobic nature, lipase substratesare not readily amenable to the modifications, such as biotinylation orother labelling, that would be required for solid phase binding.Moreover, in view of the high specificity of lipase enzymes forhydrophobic substrates, there would be no guarantee that a lipasesubstrate modified to be suitable for solid phase detection would stillbe recognised by the enzyme.

[0011] In the present invention, a lipase substrate has been modified sothat it can be bound to a solid surface, such as streptavidin-coated SPAbeads, and is shown to act as an effective lipase substrate in an assayfor lipase enzyme activity. In a particular embodiment, a substrate hasbeen designed with an amino group in the sn-2 position and oleic acid(18:1) in the sn-1 and sn-3 positions of the triacylglycerol; the oleicacid can be labelled by reductive tritiation. It has been found thatthis substrate can be biotinylated by incorporating a biotin group on aPEG spacer arm to generate a novel modified lipase substrate. Lipaseactivity removes the radiolabelled portion which, in an SPA, results ina decreased light signal from the SPA beads.

[0012] In accordance with a first aspect of the invention there isprovided a compound of

[0013] wherein L is a linking agent

[0014] B is a binding agent;

[0015] X is an atom or group suitable for attaching L to the glycerolchain; and

[0016] R is a straight chain saturated or unsaturated alkyl group havingfrom 8 to 30 carbon atoms, substituted with M′ or M″ wherein at leastone of M′ and M″ is a detectable label.

[0017] The linking agent, L, is a group which links a binding agent, B,to a triacylglycerol in such a way that the lipase substrate can beattached to a solid surface and yet still retain its biologicalactivity. Accordingly, in one embodiment of the first aspect of theinvention, the linking agent is a water-soluble molecule. This moleculecan be nontoxic and/or inert. In a preferred embodiment of the firstaspect, the linking agent, L, comprises a water-soluble compoundselected from the compounds polyethylene glycol (PEG) andpolyvinylpyrrolidone (PVP). Each of these compounds can exist as apolymer of a number of repeating units. Suitably, where L comprises PEG,the size of the PEG polymer is between 6-100 repeating units.

[0018] In another embodiment of the first aspect of the invention,binding agent, B, is capable of binding to a solid phase such as thewalls or base of wells in a plate, for example a microtitre plate, or tothe surface of a bead such as an SPA bead. Binding of the binding agent,B, to a solid surface can be direct or indirect. For example, a compoundcan be bound to a solid phase by indirect means such as by coating thesolid surface with one member of a specific binding pair and attachingthe other member to the compound of Formula I. Attachment can be bynon-covalent or covalent means.

[0019] In a preferred embodiment, binding agent, B, comprises one memberof a specific binding pair, said specific binding pair being preferablyselected from biotin:avidin or streptavidin, antibody: antigen orprotein A, receptor:ligand, nucleic acid:nucleic acid (e.g.DNA:DNA),wheatgerm agglutinin (WGA): N-acetyl β glucosamine residues orglycoproteins, glutathione:GST (glutathione-S-tranferase) andcopper:histidine tag. Other suitable specific binding pairs would beknown to those skilled in the art. Alternatively, binding can beachieved by electrostatic interaction, for example by creating apositively charged species on B which is bound to a negatively chargedspecies on the solid phase. A positive charge could be generated, forexample, using a quaternised amine.

[0020] In a particularly preferred embodiment of the first aspect of theinvention, the binding agent, B, is biotin. This would render thecompound of Formula I capable of binding to an avidin orstreptavidin-coated solid phase.

[0021] X is an atom or group which enables the linking agent, L, to bindto the carbon backbone (i.e. the glycerol chain) of the compound set outin Formula I and is chosen according to the reactive groups present onthe linking agent which are available for attachment. Thus, in oneembodiment, if the linking agent, L, has a reactive group NHS then Xwould comprise NH. In one example, the NHS group may be attached to thelinking agent through an ester linkage in which case group X maycomprise O═C—NH. In another embodiment, if the linking agent, L, hasmaleimide as a reactive group then X would comprise S. Suitable couplingmechanisms involving NH, S or O atoms would be recognised by someoneskilled in the art.

[0022] Different lipases have specificity for triacylglycerols havingfatty acids with different length carbon chains; these carbon chains aretypically between 8-30 carbon atoms and can be either saturated orunsaturated. Accordingly, in the compound of Formula I, R is a straightchain saturated or unsaturated alkyl group having from 8 and 30 carbonatoms. Stored fatty acids, such as those which form the substrates forsome lipases, generally have between 14 and 18 carbon atoms in thecarbon chain. Accordingly, in another embodiment of the first aspect ofthe invention there is provided a compound of formula I wherein R has14, 16 or 18, and preferably 18 carbon atoms.

[0023] R is substituted with M′ or M″ wherein at least one of groups M′and M″ is a detectable label. Where either of M′ or M″ is not adetectable label it would, preferably, be H. Suitable detectable labelscan be radiolabels, fluorescence labels or other labels (includingluminescent molecules for example). In one embodiment, at least one ofM′ and M″ is a radiolabel such as ³H, ¹²⁵ ^(I or) ¹⁴C, or any otherlabels which may be suitable for use in a scintillant detection systemsuch as SPA or other solid phase based assay systems (for exampleCytostar-T™ scintillating microplates (Amersham Pharmacia Biotech) orFlashplates™ (NEN)). In such systems, when the detectable label is closeto a solid surface, scintillation events can be detected but when lipaseactivity occurs the label is released with the fatty acid and thescintillant signal decreases. In a particularly preferred embodiment, M′and/or M″ is ³H. It is understood by those skilled in the art thatmigration of the fatty acid chains occurs between the sn-1, sn-2 andsn-3 positions of the carbon backbone of a triacylglycerol.

[0024] In another preferred embodiment of the first aspect of theinvention, at least one of M′ and M″ is a fluorophore. Suitablefluorophores include, for example, those based on fluorescein and itsderivatives (Handbook of Fluorescent Probes and Research Chemicals,Molecular Probes, 6^(th) Edition, 1996 or see www.probes.com) andcyanine dye molecules (Cy dyes).

[0025] In a further embodiment of the first aspect, M′ and M″ could bechosen to enable a compound of Formula I to be used in a FluorescenceResonance Energy Transfer (FRET) assay. The principal of FRET wasdescribed in U.S. Pat. No. 4,996,143 and, more recently, inPCT/GB99/01746 (publication number WO99/64519). Briefly, FRET assaysdepend on an interaction between two fluorophores, a donor fluorophoreand an acceptor fluorophore. When the donor and acceptor molecules arein close enough proximity, the fluorescence of the donor molecule istransferred to the acceptor molecule with a resultant decrease in thelifetime and a quenching of fluorescence of the donor species and aconcomitant increase in the fluorescence intensity of the acceptorspecies. When the two molecules are separated, the fluorescence of thedonor molecule is restored and the fluorescence intensity of theacceptor species decreases. The use of FRET labels in biological systemsis well known. The principle has been used in the detection of bindingevents or cleavage reactions in assays which employ FRET.

[0026] Suitable acceptor/donor pairs for use in a FRET assay aredescribed in Handbook of Fluorescent Probes and Research Chemicals,Molecular Probes (http://www.probes.com. To adapt a FRET assay for usein a lipase substrate-enzyme assay, one of the acceptor or donor wouldbe contained in the bead or other solid phase, the other would be linkedonto the substrate of Formula I in either/both the M′ and M″ position(s)such that one partner of the acceptor/donor pair was on the substrateand the other in the bead. The donor and acceptor molecules would beheld in close proximity when the substrate is bound to the bead. Uponlipase enzyme activity on the substrate, cleavage in the sn-1 and sn-3positions will cause the donor and acceptor molecules to be separatedthus restoring the fluorescence of the donor molecule with a concomitantdecrease in the fluorescence intensity of the acceptor species.

[0027] In one format of the FRET principle, a fluorescent agent iscaused to be in close proximity with a “quencher” molecule such that theenergy from the excited donor fluorophore is transferred to the quencherand dissipated as heat rather than fluorescence energy. In this case,residual fluorescence is minimised when the two components of thedonor-quencher pair are in close proximity and a large change in signalcan be obtained when they are separated.

[0028] Cyanine dyes suitable for use as acceptor or “quencher” moleculesin a FRET assay have been developed (see PCT/GB99/01746) by makingcertain modifications to cyanine dyes through introduction of chemicalgroups which have the effect of diminishing or abolishing thefluorescence of the molecule. Such quenched Cy dyes are referred to asCy-Q dyes or “dark cyanine dyes”.

[0029] Accordingly, in another embodiment of the invention, thefluorophore attached at one or both of M′ and M″ could be a “quencher”or “dark cyanine dye” molecule such as Cy-Q and the streptavidin-coatedbead could contain a normal fluorescent fluorophore. When the substrateis bound to the bead, the fluorescence of the fluorophore in the beadwill be quenched. When the lipase enzyme activity causes one or both ofthe sn-1 and sn-3 fatty acid chains to be cleaved from the substrate,the “quencher” fluorophores will be disassociated from the bead and thusthe fluorescence of the fluorophore within the bead will be restored.Accordingly, lipase enzyme activity would be detected by measuring anincrease in fluorescence.

[0030] In an alternative embodiment, the normal fluorescent fluorophorecould be attached at one or both of M′ and M″ and the “quencher”molecule could be contained in the streptavidin-coated bead.

[0031] In a second aspect of the invention, there is provided a methodfor preparing a compound according to any embodiment of the first aspectof the invention.

[0032] In one embodiment of the second aspect, the method comprises thesteps of:

[0033] a) performing a reaction to add a detectable label, M′ or M″, toa triacylglycerol; and

[0034] b) attaching a binding agent, B, to said triacylglcerol moleculethrough a linking agent, L.

[0035] In another embodiment, step a) comprises a tritiation reaction,preferably adding ³H to the acyl carbon chains in the sn-1 and/or sn-3positions.

[0036] In a further embodiment, B is attached to a triacylglycerolmolecule via L wherein L is a PEG spacer arm. In a particularlypreferred embodiment, step b) comprises reacting said triacylglycerolmolecule with Biotin PEG NHS ester.

[0037] The compound of Formula I forms a substrate for a lipase. Lipaseswhich can be assayed with a substrate of Formula I include lipasesextracted from mammalian, yeast or bacterial cells (examples of lipasesinclude those extracted from wheat germ, Chromobacterium, Mucor,Pseudomonas, Candida, lipoprotein lipase, hepatic and pancreatic lipasesand Hormone Sensitive Lipase (HSL)). A composition can comprise asolution containing a lipase enzyme or a cell or cell extract. Inprinciple, any type of cell can be used i.e. prokaryotic or eukaryotic(including bacterial, mammalian and plant cells). Where appropriate, acell extract can be prepared from a cell, using standard methods knownto those skilled in the art (Molecular Cloning, A Laboratory Manual2^(nd) Edition, Cold Spring Harbour Laboratory Press 1989).

[0038] In a third aspect of the invention there is provided an assay fordetecting lipase enzyme activity in a composition, said assay comprisingincubating said composition in the presence of the compound of Formula Ias claimed in any embodiment of the first aspect and measuring releaseof the detectable label, M′ and/or M″, from the compound as anindication of lipase activity.

[0039] The presence of binding agent, B, in the compound of Formula Imakes it possible to use this enzyme substrate in a homogeneous assaywhich involves a solid phase binding step.

[0040] Accordingly, in a fourth aspect of the invention there isprovided an assay for detecting lipase enzyme activity in a compositioncomprising:

[0041] a) mixing said composition with a compound of Formula I asclaimed in any embodiment of the first aspect;

[0042] b) incubating under conditions to promote lipase enzyme activity,

[0043] c) adding a solid phase under conditions to promote binding ofthe compound to the solid phase; and

[0044] d) detecting the amount of M′ and/or M″ on the solid phase as anindication of lipase enzyme activity.

[0045] In a fifth aspect of the invention there is provided an assay fordetecting lipase enzyme activity in a composition comprising:

[0046] a) incubating the compound of Formula I as claimed in anyembodiment of the first aspect with a solid phase under conditions topromote solid phase binding;

[0047] b) adding said composition to said solid phase-bound compound;

[0048] c) incubating under conditions to promote lipase enzyme activity;

[0049] e) detecting the amount of M′ and/or M″ on the solid phase as anindication of lipase enzyme activity.

[0050] In one embodiment of the fourth or fifth aspects binding of thecompound to the solid phase brings the compound into proximity with ascintillant and detection of M′ and/or M″ on the solid phase is bycounting scintillation events i.e. “scintillation counting”.

[0051] In another embodiment of the fourth or fifth aspects the solidphase is the surface of an SPA bead, preferably a streptavidin coatedyttrium silicate (YSi) or PVT (poly (vinyl toluene)) SPA bead. Othersuitable beads include or YOx (yttrium Oxide) (Amersham PharmaciaBiotech) or polystyrene (PST) beads.

[0052] In a further embodiment there is provided an assay wherein thesolid phase is a surface of a plate such as a microtitre plate,preferably a Cytostar™ plate (Amersham Pharmacia Biotech) orFlashplates™ (NEN).

[0053] Enzyme assays can, for example, be run in 96 SPA or 384 wellLEADseeker™ format and will be suitable for use in screening. TheLEADseeker™ system features a CCD camera allowing imaging of highdensity microtitre plates in a single pass. This can be used for readingassays in radioactive, fluorescent and luminescent formats.

[0054] The lipase enzyme substrate of Formula I can be used in an assayfor identifying the presence in a composition of a compound which canmodify lipase enzyme activity. Accordingly, in a particularly preferredembodiment of any of the third, fourth or fifth aspects of the inventionsaid composition comprises a lipase enzyme and a putative inhibitor orenhancer of lipase enzyme activity.

[0055] In one embodiment of any of the third, fourth or fifth aspects ofthe invention, the assay further comprises adding a stop solution to thereaction mixture prior to measuring M′ or M″.

[0056] In a sixth aspect of the invention there is provided a use of acompound of Formula I in accordance with any embodiment of the firstaspect in an assay according to any embodiment of the third, fourth orfifth embodiments.

[0057] The present invention is further illustrated with reference tothe following Figures and Examples in which:

[0058]FIG. 1 shows a reaction Scheme 1 for preparation of HSL substrate.

[0059]FIG. 2 shows the results of an enzyme titration comparing ‘on’ (∘)and ‘off’ (•) bead assay formats.

[0060]FIG. 3 shows the effect of time on substrate cleavage by 5 nMlipase.

[0061]FIG. 4 shows inhibition of lipase activity by Ebelactone B.

[0062]FIG. 5 shows the results of an enzyme titration comparing SPA (∘)and LEADseeker (•) assay formats.

[0063]FIG. 6 shows inhibition of lipase activity by Ebelactone Bdetected using YSi SPA beads (∘) and YOx LEADseeker beads (•).

[0064]FIG. 7 demonstrates cleavage of HSL substrate by a range oflipases.

[0065]FIG. 8 shows inhibition of HSL by PMSF.

EXAMPLE 1

[0066] A method for synthesising a lipase substrate for HSL is set outin Reaction Scheme 1 (see FIG. 1).

[0067] Briefly, a tritiation reaction was carried out as follows:

[0068] Approximately 25 mg of the hydrochloride salt of9(Z)-octadec-9-enoic acid 2-amino-3-3((9Z)-octadec-9-enoyloxy)propylester (NNC 90-3086) was combined with 10% palladium on charcoal (25 mg)and methanol (1.5 ml) in a tritiation vessel and stirred under tritiumgas (10 Ci) for 90 minutes. This gave a crude yield of tritiated productof approximately 3.3 Ci.

[0069] This crude material was purified by high performance liquidchromatography (HPLC) on a Progidy ODS column (Phenomenex) using amethanol:water:trifluroacetic acid (90:10:0.1) (buffer A) andmethanol:trifluroacetic acid (100:0.1) (buffer B). A gradient of 0% B to100% B was run over 30 minutes at 3 ml/min. The yield after purificationwas approximately 1.6 Ci.

[0070] The tritiated compound, [³H]NNC 90-3086 (1.6 Ci, 2.5 ml), wasthen biotinylated by forming a dimethylformamide solution and adding itto a Biotin PEG NHS ester (from Shearwater Polymers, Inc.) (MW˜3400, 57mg) and diisopropylethylamine (100 μl). The reaction mixture was heatedat 100° C. for 45 minutes.

[0071] The resultant product was partially purified on a short silicacolumn eluting in dichloromethane:methanol (9:1) then purified by thinlayer chromatography on a short silica gel eluting in 4 mldichloromethane followed by 8 ml dichloromethoane:methanol (9:1).

[0072] Typically, the radiochemical purity of [³H]HSL substrate achievedwas >95% and the specific activity was ˜100 Ci/mmol.

EXAMPLE 2

[0073] A Screening Assay for a Lipase Using Scintillation and ImagingProximity Assay Technologies.

[0074] Method

[0075] 96-well SPA format. Using an ‘on bead’ assay format, 180 nMlipase substrate (i.e. HSL substrate prepared by the method described inExample 1) was added to streptavidin coated yttrium silicate (YSi) beads(Amersham Pharmacia Biotech) at 5 mg/ml and 10% (v/v) Triton™ X-100 inthe ratio of 1:3:2. Triton X-100 was added to prevent non-specificbinding of the tritiated product to beads. Assays contained 20 μlsubstrate precoated SPA beads (6 nM lipase substrate, 50 μg beads, 0.67%(v/v) Triton X-100), 5 nM lipase and assay buffer (50 mM Hepes, pH 7.5,1 mM dithioerythritol (DTE) and 0.001% (v/v) C13E12) in a volume of 100μl. In ‘no enzyme’ controls, approximately 15000 SPA cpm were obtained.After incubation for 60 minutes at room temperature with agitation,assays were stopped by the addition of 100 μl 0.1M sodium citrate/citricacid, pH 4.0 and counted on a TopCount™ microplate scintillation counter(Packard Instruments Co., Meriden, Conn., USA).

[0076] 384-well SPA and LEADseeker formats. 180 nM lipase substrate wasadded to streptavidin coated YSi beads at 10 mg/ml and 10% (v/v) Triton™X-100 in the ratio of 2:3:1. Assays contained 101 μl substrate precoatedstreptavidin YSi SPA beads or streptavidin yttrium oxide (YOx)LEADseeker beads (Amersham Pharmacia Biotech) (24 nM lipase substrate,50 μg beads, 0.67% (v/v) Triton X-100), 30 nM lipase and assay buffer ina volume of 25 μl. In ‘no enzyme’ controls, approximately 6000 SPA cpmand 600 IOD's (Integrated Optical Density) were obtained. Afterincubation for 60 minutes at room temperature with agitation, assayswere stopped by the addition of 25 μl 0.1M sodium citrate/citric acid,pH 4.0 and counted on either on a TopCount microplate scintillationcounter (Packard Instruments Co., Meriden, Conn., USA) or imaged onLEADseeker (Amersham Pharmacia Biotech).

[0077] Results

[0078] Development of a lipase assay using a 96-well SPA assay format.Enzyme titrations were set up using ‘on’ and ‘off’ bead assay formats.In the ‘off’ bead format, the lipase substrate was not precoated ontoSPA beads; instead the beads were added at the same time as the stopsolution. Using an ‘on’ bead assay format resulted in a higherpercentage of substrate cleavage than using an ‘off’ bead format and amore reproducible assay, as seen by the tighter error bars. It isthought that the binding of the substrate to the bead surface producedan artificial membrane environment necessary for optimal enzyme activity(FIG. 2). Each data point in this and subsequent figures is the mean(±SEM) of 3 replicates.

[0079] The maximum substrate cleavage obtained was approximately 60%.Due to the nature of the ‘on’ bead assay, it is unlikely that 100%substrate cleavage would be obtained since steric hindrance, caused bythe presence of the bead, is likely to affect enzyme activity.

[0080] Using the ‘on’ bead assay format, a time course was establishedusing 5 nM lipase (FIG. 3).

[0081] Ebelactone B, a natural product from Streptomyces aburaviensis,is known to be an inhibitor of lipase activity (Nonaka, Y. et al, J.Enzyme Inhibition 10, pp 57-63 (1995)). Inhibition studies were carriedout using Ebelactone B and an IC₅₀ value of 27.5 μM was obtained (FIG.4). 5 nM lipase was used and assays were incubated at room temperaturefor 60 minutes.

[0082] Miniaturization of the lipase assay to 384-well format using SPAand LEADseeker. Using a reduced assay volume an enzyme titration wasperformed with YSi SPA beads and YOx LEADseeker beads (FIG. 5).Different profiles were obtained using the two assay formats and thismay be a result of how the substrate is presented on the surface of thetwo bead types.

[0083] Inhibition curves were obtained for the miniaturized assays usingEbelactone B and IC₅₀ values of 32.3 μM and 18.2 μM were determinedusing SPA and LEADseeker formats, respectively (FIG. 6). These valuesare comparable to the one calculated using the 96-well SPA assay format.30 nM lipase was used and assays were incubated at room temperature for60 minutes.

EXAMPLE 3

[0084] Cleavage of Lipase Substrate by Other Lipases

[0085] The standard assay format (as described in Example 2 above) wasperformed with lipase enzymes derived from number of different sources.The results are shown in FIG. 7.

EXAMPLE 4

[0086] Inhibition of HSL Activity by PMSF

[0087] The inhibition of HSL activity by PMSF was investigated using thestandard assay format (as described above in Example 2) and 14.35 mM to1.44 nM PMSF. Assays were incubated at room temperature for 60 minutes.

[0088] An IC₅₀ value of 16.3 μM was obtained. In similar experiments,IC₅₀ values of 4.8, 10.6 and 16.7 μM were obtained. Results are shown inFIG. 8.

What is claimed is:
 1. (once amended) A compound comprising Formula I:

wherein L is a linking agent including a water-soluble molecule; B is abinding agent; X is an atom or group suitable for attaching L to aglycerol chain; and R is a straight chain saturated or unsaturated alkylgroup having from 8 to 30 carbon atoms, substituted with M′ or M″wherein at least one of M′ and/or M″ is a detectable label.
 3. (onceamended) The compound of claim 1 wherein L includes a water-solublemolecule selected from PEG or PVP.
 4. (once amended) The compound ofclaim 1 wherein L includes 6-100 units of PEG.
 5. (once amended) Thecompound of claim 1 wherein B is capable of binding to a solid phase. 6.(once amended) The compound of claim 1 wherein B includes one member ofa specific binding pair.
 7. (once amended) The compound of claim 1wherein B is biotin.
 8. (once amended) The compound of claim 1 wherein Xis NH, O or S.
 9. (once amended) The compound of claim 1 wherein R has14, 16 or 18 carbon atoms.
 10. (once amended) The compound of claim 1wherein at least one of M′ and M″ is a radioactive label.
 11. (onceamended) The compound of claim 1 wherein at least one of M′ and M″ is afluorophore.
 13. (once amended) A method for preparing the compound ofclaim 1 comprising the steps of a) performing a reaction to add adetectable label, M′ or M″, to a triacylglycerol; and b) attaching abinding agent, B, to said triacylglcerol molecule through a linkingagent, L.
 14. (once amended) The method of claim 13 wherein step a)includes a tritiation reaction, preferably adding ³H in the sn-1 and/orsn-3 positions.
 15. (once amended) The method of claim 13 wherein stepb) includes reacting said triacylglycerol molecule with Biotin PEG NHSester.
 16. (once amended) An assay for detecting lipase enzyme activityin a composition comprising incubating said composition in the presenceof the compound of claim 1 and measuring release of the detectablelabel, M′ and/or M″, from the compound as an indication of lipaseactivity.
 17. (once amended) An assay for detecting lipase enzymeactivity in a composition comprising: a) mixing said composition withthe compound of claim 1; b) incubating under conditions to promotelipase enzyme activity; c) adding a solid phase under conditions topromote binding of the compound to the solid phase; and d) detecting theamount of M′ and/or M″ on the solid phase as an indication of lipaseenzyme activity.
 18. (once amended) An assay for detecting lipase enzymeactivity in a composition comprising: a) incubating the compound ofclaim 1 with a solid phase under conditions to promote solid phasebinding; b) adding said composition to said solid phase-bound compound;c) incubating under conditions to promote lipase enzyme activity; d)detecting the amount of M′ or M″ on the solid phase as an indication oflipase enzyme activity.
 19. (once amended) The assay of claim 17 whereinbinding to the solid phase brings the compound into proximity with ascintillant and detection of M′ or M″ on the solid phase is by measuringscintillation counts.
 20. (once amended) The assay of claim 17 whereinthe solid phase is the surface of an SPA bead, preferably a streptavidincoated yttrium silicate SPA bead.
 21. (once amended) The assay of claim17 wherein the solid phase is a surface of a plate.
 22. (once amended)The assay of claim 16 wherein said composition comprises a lipase enzymeand a putative inhibitor or enhancer of lipase enzyme activity. 23.(once amended) The assay of claim 16 wherein a stop solution is addedprior to detecting M′ and/or M″.
 25. (new) The compound of claim 6wherein the specific binding pair is selected from the group consistingof biotin:avidin/streptavidin, antibody:antigen/protein A, nucleicacid:nucleic acid, receptor:ligand, WGA:N-acetyl β glucosamine,glutathione:GST (glutathione-S-transferase) and copper:histidine tag.26. (new) The assay of claim 21 wherein the plate is a microtitre plate.